利用阿拉伯芥 T-DNA Knockout 突變株 , 研究調控葉部形態發育之功能性基因(3/3)

Abstract

Leaves are the most noticeable plant organs and play important roles in photosynthesis, respiration and gas exchange. However, the details of leaf development remain unclear. Therefore, our laboratory used forward genetics to identify the mutants with leaf development defect from T-DNA tagging pools in Arabidopsis. We use a T-DNA tagging vector, pPZP202: BAR: SK, to generate mutants and to screen for morphological mutants. In this study, we have identified a mutant, R1-2, which were selected in the T2 generation, with curled leaves and delayed flowering phenotypes. After R1-2 was crossed to the WT, none of the R1-2 phenotypes was observed in the F1 but were found in the F2 population. Therefore, it was suggested that R1-2 was a recessive mutant. Southern blotting analysis and inversed PCR identified two T-DNA linked together and inserted in the 2.5kb upstream of the At3g22970. Using RT-PCR and Northern blotting assay, we showed that the transcripts of At3g22970 were down-regulated in mutant, implicating that the phenotypes of R1-2 may be resulted from down-regulation of At3g22970. At3g22970 was predicted as a protein containing DUF506 domain and located in the chloroplast. There are 14 genes that contain DUF506 domain in Arabidopsis thaliana and all with unknown function. Using RT-PCR and Northern blotting assay, it was detected that the expression of At3g22970 appeared rhythmic, and expressed in the whole plant, especially in flowers. However, the function of At3g22970 is still unknown. We observe the phenotype of the transgenic plants by over-expressing or knock-outing the At3g22970 to address its function in Arabidopsis. Among these over-expression lines, some lines with delayed flowering phenotype had reduced expression of At3g22970, but others were the same as WT. The correlation between delayed flowering time phenotype and reduced expression of At3g22970, indicating that the phenotype was caused by co-suppression of the over-expressed At3g22970. Additionally, one of the SALK knock-out lines, SALK-085910, also exhibited a delayed flowering phenotype. Therefore, At3g22970 might play a role to control flowering time.